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Development of a RP-HPLC Method for Separating and Quantifying Muscimol in Different Developmental Stages of the Fungus Amanita Muscaria

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Development of a RP-HPLC Method for Separating and Quantifying Muscimol in Different Developmental Stages of the Fungus Amanita Muscaria Hindawi Journal of Chemistry Volume 2020, Article ID 8859998, 4 pages https://doi.org/10.1155/2020/8859998 Research Article Development of a RP-HPLC Method for Separating and Quantifying Muscimol in Different Developmental Stages of the Fungus Amanita muscaria Emerso´n Leo´n A´ vila and James Guevara-Pulido Universidad El Bosque, Qu´ımica Farmace´utica, INQA Research Group, Av Cra 9 No. 131A-02, Bogota´ 110121, Colombia Correspondence should be addressed to James Guevara-Pulido; [email protected] Received 25 August 2020; Accepted 4 November 2020; Published 20 November 2020 Academic Editor: Murat Senturk Copyright © 2020 Emers´on Le´on A´ vila and James Guevara-Pulido. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A simple RP-HPLC method was designed for the quantification of muscimol (5-(aminomethyl)-isoxazol-3-ol) present in five aqueous extracts of Amanita muscaria each from a different developmental stage. Results show that the maximum concentration of muscimol (1,210 mg/ml) was found in the young mushroom stage of development. Moreover, it was also found that this concentration progressively decreases as the fungus ages. -e developed method is a simple but effective method for the quantification of muscimol, a widely important metabolite for the pharmaceutical industry as a possible treatment for tardive dyskinesia and Parkinson’s disease. 1. Introduction HPLC specifically has been described as a reliable tool for the identification and quantification of MUS and IBO. One Amanita muscaria, commonly known as “fly agaric” or “fly study quantified both analytes by derivatizing them with dansyl amanita”, is one of the best known psychoactive mushrooms chloride (DNS-Cl) [9], while another study described a method in the world due to its psychotropic properties [1]. -is based on the interaction of sensitive ions which allowed for the fungus is distinguished by a bright red cap featuring small simultaneous detection of IBO and MUS [10]. white warty spots, and it is generally found in Europe, Africa, Considering that most available methods for the Asia, and the Americas. In Colombia, it was introduced as a quantification of muscimol require a chemical transfor- symbiont with pine and eucalyptus trees [2]. -e main mation of the analyte, the following study proposes a simple psychoactive constituents of this basidiomycete are neuro- derivatization-free, RP-HPLC-based method that allows for toxins ibotenic acid (IBO) and muscimol (MUS), both of the quantification of muscimol (MUS) in aqueous extracts of which are of interest due to their hallucinogenic and different developmental stages of A. muscaria. Additionally, pharmacological properties [3]. Currently, medical research this method provides a way of identifying which develop- has focused on the use of MUS as GABAA receptor agonists mental stage of A. muscaria will yield the highest concen- as a possible treatment for tardive dyskinesia and Parkin- tration of MUS in order for it to be used as a GABAA son’s disease [4]. receptor agonist. Several studies have sought to identify and quantify various compounds present in this fungus such as musca- 2. Experimental rine, bufotenin, muscimol (Figure 1), ibotenic acid [5], muscazone, amatoxins, and phallotoxins [6] by use of an- 2.1. Muscimol Extraction. A standard sample of solid alytical techniques including paper chromatography [7], muscimol and Sigma HPLC grade methanol (St. Louis, MO, zone capillary electrophoresis, HPLC, and GC/MS [8]. USA), as the solvent for the mobile phase, were acquired via 2 Journal of Chemistry OH OH CO OH 2 H N 2 N N H N O H N 2N 2 O O O OH Figure 1: Muscimol (5-(aminomethyl)-isoxazol-3-ol). IBO MUS Figure 2: IBO transformation into MUS through drying-mediated decarboxylation [3]. “Suministros Analiticos S.A.A de Colombia” as Toronto Research Chemicals distributor. For this study, an array of Amanita muscaria samples approach for the separation of the matrix. -is can be were collected in Bogota-Colombia (lat-long coordinates: explained by the fact that, according to Meyer, v. R., the 4.707953, −74.026984; altitude: 2577.694 m.a.s.l), which retention factor (k′) in RP-HPLC decreases when the were then classified according to their developmental stage: organic portion of the mobile phase is increased [13]. On button stage, young mushroom stage, and mature mush- the other hand, increasing the water content of the room stage [11]. mobile phase increased the retention time and allowed First, samples were dried in order to guarantee their for the separation of the MUS peak, which was contrasted quality, avoid microbial contamination, and promote the with, and then confirmed by the peak from the MUS decarboxylation of IBO to obtain MUS [12], as shown in standard solution. Consequently, it was established Figure 2. that the best mobile phase was constituted by a 49/1 ratio -en, samples were lyophilized and mechanically frag- of H2O/MeOH (i.e., using 2% of MeOH and the rest mented to promote the liquid extraction of muscimol using water). water as the solvent, which yielded a complex matrix of MUS Now, in order to use the least amount of mobile phase and other compounds. Finally, a sonication pretreatment while decreasing the retention times in an effort to make the was carried out before extraction via HPLC. quantification method more efficient, the ratio of the mobile phase was maintained throughout, but the flow rate was adjusted by 0.1 mL/min from 0.100 mL/min to 2.000 mL/ 2.2. Muscimol Separation from the Matrix. Muscimol is a min (0.100 mL/min, 0.200 mL/min, 0.300 mL/min, polar molecule that exhibits high solubility in water; 0.400 mL/min, etc). After examining the resultant chro- therefore, the most suitable technique to work with was matograms, it was determined that greater flow rate results reversed-phase chromatography (RP-HPLC). in shorter retention times but also results in overlapping -e first trials were carried out with a standard solution peaks and an inferior symmetry of the MUS standard so- of MUS and an aqueous extract of A. muscaria obtained lution peak. -erefore, it was concluded that the best-suited from approximately 1 g of lyophilized fungus and 10 mL of flow rate is 0.10 mL/min. All trials were carried out with an water as described in the previous section. Retention times, injection volume of 10 μL, and no widening of the standard peak symmetry, repeatability, and reproducibility were peak was observed. analyzed in order to understand the complexity of the matrix. Considering that the retention time using a C18 column 2.3. Calibration Curve. Once the most favorable chro- was appropriate, that at 256 nm the analyte was well- matographic conditions were determined for MUS sepa- detected, and that the time/area showed good reproduc- ration, solutions made up of different concentrations of the ibility, it was deemed that the method could be designed standard MUS solution (1.54, 1.00, 0.77, 0.4620, 0.3080, evaluating only variables like flow rate and polarity of the 0.154, 0.0770, 0.0462, and 0.0304 mg/mL) were injected into mobile phase. a Shimadzu HPLC (Japan, Prominence-i model LC 2030) at -us, trials to determine the best mobile phase were 256 nm, with an Ultra Aqueous (AQ) C18 column and a performed. Given that the polarity of the functional groups water-methanol (98 : 2) mobile phase at a flow rate of 0.1 mL/ present in the analytes determines the elution order of the min. Subsequently, a calibration curve was plotted compounds and recognizing the presence of hydroxy and (Figure 3), and areas under the curve were calculated. amino groups in MUS, the retention times were expected to Overall, five series of each standard solution were analyzed be short. Since the elution order can be modified by in order to verify reproducibility and to carry out a statistical changing the polarity of the mobile phase, the proportion analysis of the results. of water to “organic modifier” (methanol) was adjusted, to According to Figure 3, the calibration curve appears find the optimum polarity of this phase in regard to the adequate for estimating the concentrations of the com- retention factor (k′) as such: H2O/MeOH: 3/1, 3.5/1, 4/1, pounds present in the matrix since it shows linearity which, 19/1, 49/1. in turn, suggests that the method is reliable in its ability to However, increasing the concentration of methanol obtain directly proportional results with the analyte con- decreases the retention time of the whole sample indi- centration in the solutions for the quantification of mus- cating an overlap of peaks, which suggests that increasing cimol at the previously mentioned developmental stages of the organic portion of the mobile phase is an infeasible Amanita muscaria. Journal of Chemistry 3 90000000 Detector A channel 2.256nm 150 80000000 70000000 5,622 60000000 100 50000000 4,041 40000000 Area 30000000 50 3,470 20000000 10,503 y = 5E + 07x – 44714 10000000 R2 = 0.9979 14,657 0 0 2,170 –10000000 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0.0 2.5 5.0 7.5 10.0 12.5 15.0 Concentration of MUS mg/mL Min Figure Figure 3: Calibration curve of muscimol. 4: Muscimol chromatogram from Amanita muscaria samples also featuring the standard sample of MUS. 3. Results and Discussion Table 1: MUS concentration in samples of the developmental stages of the fungus. After determining the most favorable chromatographic Sample Muscimol Muscimol conditions and building the calibration curve, solutions of Mass number concentration recovery the samples of fungi were injected into the RP-HPLC.
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